title: Silibinin as a potential therapeutic for sulfur mustard injuries.
abstract: Sulfur mustard (SM) is a vesicating chemical warfare agent causing skin blistering, ulceration, impaired wound healing, prolonged hospitalization and permanent lesions. Silibinin, the lead compound from Silybum marianum, has also been discussed as a potential antidote to SM poisoning. However, its efficacy has been demonstrated only with regard to nitrogen mustards. Moreover, there are no data on the efficacy of the water-soluble prodrug silibinin-bis-succinat (silibinin-BS). We investigated the effect of SIL-BS treatment against SM toxicity in HaCaT cells with regard to potential reduction of necrosis, apoptosis and inflammation including dose-dependency of any protective effects. We also demonstrated the biotransformation of the prodrug into free silibinin. HaCaT cells were exposed to SM (30, 100, and 300μM) for 30min and treated thereafter with SIL-BS (10, 50, and 100μM) for 24h. Necrosis and apoptosis were quantified using the ToxiLight BioAssay and the nucleosome ELISA (CDDE). Pro-inflammatory interleukins-6 and -8 were determined by ELISA. HaCaT cells, incubated with silibinin-BS were lysed and investigated by LC-ESI MS/MS. LC-ESI MS/MS results suggest that SIL-BS is absorbed by HaCaT cells and biotransformed into free silibinin. SIL-BS dose-dependently reduced SM cytotoxicity, even after 300μM exposure. Doses of 50-100μM silibinin-BS were required for significant protection. Apoptosis and interleukin production remained largely unchanged by 10-50μM silibinin-BS but increased after 100μM treatment. Observed reductions of SM cytotoxicity by post-exposure treatment with SIL-BS suggest this as a promising approach for treatment of SM injuries. While 100μM SIL-BS is most effective to reduce necrosis, 50μM may be safer to avoid pro-inflammatory effects. Pro-apoptotic effects after high doses of SIL-BS are in agreement with findings in literature and might even be useful to eliminate cells irreversibly damaged by SM. Further investigations will focus on the protective mechanism of silibinin and its prodrug and should establish an optimum concentration for treatment.
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany. Electronic address: frankbalszuweit@bundeswehr.org.
authors.forename: Frank
authors.initials: F
authors.lastname: Balszuweit
authors.forename: Harald
authors.initials: H
authors.lastname: John
authors.forename: Annette
authors.initials: A
authors.lastname: Schmidt
authors.forename: Kai
authors.initials: K
authors.lastname: Kehe
authors.forename: Horst
authors.initials: H
authors.lastname: Thiermann
authors.forename: Dirk
authors.initials: D
authors.lastname: Steinritz
mesh_terms: D000931:Antidotes / Q000493:pharmacokinetics / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D001711:Biotransformation; D002460:Cell Line; D002619:Chemical Warfare Agents / Q000633:toxicity*; D019610:Cytoprotection / Q000187:drug effects; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis; D016209:Interleukin-8 / Q000096:biosynthesis; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009151:Mustard Gas / Q000633:toxicity*; D009336:Necrosis; D000077385:Silybin; D012838:Silymarin / Q000493:pharmacokinetics / Q000627:therapeutic use*; D012867:Skin / Q000187:drug effects* / Q000293:injuries*; D023382:Toxicity Tests, Acute
chemical_list: D000931:Antidotes; C508606:CXCL8 protein, human; D002619:Chemical Warfare Agents; C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D012838:Silymarin; D000077385:Silybin; D009151:Mustard Gas
keywords: Biotransformation; Cell culture model; Cytotoxicity; Inflammation; Silibinin; Sulfur mustard